1. Field of the Invention
The present invention relates generally to the fields of pharmaceutical sciences, protein chemistry, polymer chemistry, colloid chemistry, immunology, and biomedical engineering. More specifically, the present invention relates to a novel delivery system for vascularization agents and other growth factors and drugs.
2. Description of the Related Art
Microparticulate systems are particles having diameter 1-2,000 xcexcm (2 mm), more preferably 100-500 xcexcm (microcapsules). Nanoparticles range from 1-1000 nm (1 xcexcm=1,000 nm), preferably 10-300 nm. Alternatively, polymeric films of 0.5 to 5-mm thickness can be made. Also, absorbable or nonabsorbable polymers can be coated with a polymeric film. Collectively, these systems will be denoted as drug delivery vehicles. All these vehicles can be formed from variety of materials, including synthetic polymers and biopolymers (proteins and polysaccharides) and can be used as carriers for drugs and other biotechnology products, such as growth factors and genes.
In the scientific realm of controlled drug delivery, the drug delivery vehicles are formed in a mixture with the agent to be encapsulated for subsequent sustained release. A number of different techniques are used to make these vehicles from synthetic or natural polymers. These techniques include phase separation, precipitation, solvent evaporation, emulsification, spray drying, casting of polymers into a polymeric sheet, or any combination thereof [Desay, P. B. Microencapsulation of drugs by pan and air suspension technique. Crit. Rev. Therapeut. Drug Carrier Syst., 5: 99-139 (1988); Berthold, A., Cremer, K., Kreuter, J. Preparation and characterization of chitosan microspheres as drug carrier for prednisolone sodium phosphate as model antiinflammatory drugs. J. Controlled Release 39: 17-25 (1996); Watts, P. J., Davies, H. C., Melia, C. D. Microencapsulation using emulsification/solvent evaporation: An overview of techniques and applications. Crit. Rev. Therapeut. Drug Carrier Syst. 7: 235-159 (1990); Cowsar, D. R., Tice, T. R., Gilley, R. M., English, J. P. Poly(lactide-co-glycolide) microcapsules for controlled release of steroids. Methods Enzymol. 112: 101-116 (1985); Genta, I., Pavanetto, F., Conti, B., Ginnoledi, P., Conte, U. Spray-drying for the preparation of chitosan microspheres. Proc. Int. Symp. Controlled Release Mater. 21: 616-617 (1994)].
Polymeric vehicles can be prepared either from preformed polymers, such as polylactic acid, polylactic-glycolic acid [Cohen, S., Yoshioka, T., Lucarolli, M., Hwang, L. H., Langer, R. Controlled delivery systems for proteins based on poly(lactic/glycolic acid) microspheres. Pharm. Res. 8: 713-720 (1991)], or from a monomer during polymerization, such as polyalkylcyanoacrylates [Al-Khouri-Fallouh, N., Roblet-Trempel, L., Fessi, M, Devissaguet, J.-P., Puisieux, F. Development of new process for the manufacture of polyisobutylcyanoacrylate nanoparticles. Int. J. Pharm. 28: 125-132 (1986)]. Both of these technologies have limited application due to the use of organic solvents, which leave residual organic solvents in the final product. Although the polyalkylcyanoacrylate nanoparticulate technology is also available in a water-based system [Couvreur, P., Roland, M., Speiser, P. Biodegradable submicroscopic particles containing a biologically active substance and compositions containing them. U.S. Pat. No. 4,329,332 (1982)], animal studies demonstrated the presence of toxic degradation products [Cruz, T., Gaspar, R., Donato, A., Lopes, C. Interaction between polyalkylcyanoacrylate nanoparticles and peritoneal macrophages: MTT metabolism, NBT reduction, and NO production. Pharm. Res. 14: 73-79 (1997)].
Cell encapsulation [Chang, T. M. Hybrid artificial cells: Microencapsulation of living cells. ASAIO Journal 38: 128-130 (1992)] is a related technology that has also been explored for the purpose of making micro- and nanoparticles. Such particles can be formed either by polymer precipitation, following the addition of a non-solvent or by gelling, following the addition of a small inorganic ion (salt) and a complexing polymer (of an opposite charge). If a long enough time is allowed the particle interior (core) can be completely gelled. Usually, the inner core material is of a polyanionic nature (negatively charged polymer). The particle membrane (shell) is made from a combination of polycation (positively charged polymer) and polyanion. The core material is usually atomized (nebulized) into small droplets and collected in a receiving bath containing a polycationic polymer solution. The reciprocal structure is also possible. In this scenario, core material is polycationic and the receiving bath is polyanionic. Several binary polymeric encapsulation systems (resulting from two polymers) have been described [Prokop, A., Hunkeler, D., DiMari, S., Haralson, M. A., Wang, T. G. Water soluble polymers for immunoisolation. I. Complex caocervation and cytotoxicity. Advances in Polymer Science, 136: 1-51 (1998)]. These systems are inadequate due to the fact that the membrane parameters are governed by a single chemical complex resulting from the ionic interactions. The inability to adjust independently particle parameters hinders the success of these systems.
In an effort to overcome these severe limitations, new multicomponent polymeric micro- and nanoparticles were designed that permit independent modification of mechanical strength and permeability [Prokop, A., Hunkeler, D., Powers, A. C., Whitesell, R. R., Wang, T. G. Water soluble polymers for immunoisolation. II. Evaluation of multicomponent microencapsulation systems. Advances in Polymer Science, 136: 52-73 (1998)]. Over one thousand combinations of polyanions and polycations were examined as polymer candidates suitable for encapsulation of living cells. Thirty-three combinations were found to be usable. However, microcapsules are not always best suited as delivery vehicles because of their relatively large size. In addition, the composition and concentrations claimed in [Wang, T. G., Lacik, I., Brissova, M., Anilkumar, A. V., Prokop, A., Powers, A. C. Encapsulation system for the immunoisolation of living cells. U.S. Pat. No. 599,790, 1997] do not allow for the generation of small nanoparticles, suitable for injectable drug delivery. Such system has recently been described in a patent application [Prokop, A.: Micro- and nano-particulate polymeric delivery system, U.S. patent application, 1997].
Diabetes is a chronic disease, characterized by a high morbidity and mortality rate due to major complications (blindness, renal failure, and neuropathy) [The Diabetes Control and Complications Trial Research Group (DCCT). The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin dependent diabetes mellitus, New England J. Med. 329: 977-986 (1993)]. During the past 10 years it has become clear that none of the alternative treatment strategies (such as gene therapy, islet transplantation, beta cell bioartificial pancreas) provide a sufficient benefit to risk ratio. It is, nevertheless, difficult using current technologies to maintain normal blood sugar levels in individuals with diabetes. Considerable research has been devoted to the development of alternative methods for reestablishing normoglycemia. Pancreas and islet transplantation results have been disappointing, and the procedures are unlikely to receive widespread use [Lacy, P. E. Islet transplantationxe2x80x94The future, in: Pancreatic Islet Cell Transplantation, Ricordi, C., ed., R. G. Landes, Austin, pp. 394-399, 1992; Mintz, D. H. and Alejandro, R. Islet cell transplantation, In: Pancreatic Islet Cell Transplantation, Ricordi C, ed., R. G. Landes, Austin, pp. 1-6, 1992]. The use of non-human islets is limited by their immediate rejection and destruction by the recipient unless a potent immune suppressive therapy is applied. An ideal treatment for diabetes would isolate the donor animal islets from the host immune system while allowing other islet functions such as metabolism and insulin production to proceed without restriction.
Encapsulation of living islets from animals may overcome the problems facing the whole-organ transplantation. Encapsulating the living islets in a protective membrane or microcapsules would allow insulin to be secreted, yet prevent the immune system from rejecting the islet [Lim, F. and Sun, A. M. Microencapsulated islets as bioartificial endocrine pancreas, Science 210: 908-910 (1989)]. The recipient would not need to be placed on anti-rejection drug regimen as currently practiced in whole-organ transplantation. Since these drugs themselves pose significant danger to the patient, the advantages of transplanting encapsulated islets are evident.
A number of encapsulation processes are available today, however, none of these methods fulfill the requirements for transplantation [Calafiore, R. The large-scale microencapsulation of isolated and purified human islets of Langerhans, In: Pancreatic Islet Cell Transplantation, Ricordi C, ed., R. G. Landes, Austin, pp. 207-214, 1992; Lanza, R. P., Sullivan, S. J., Monaco, A. P. and Chick, W. L. The hybrid artificial pancreas: Diffusion and vascular devices, In: Pancreatic Islet Cell Transplantation, Ricordi C, ed., R. G. Landes, Austin, pp. 223-237, 1992; Soon-Shiong, P., Heitz, R. E., Merideth, N., Yao, Q. X., Yao, Z., Zheng, T., Murphy, M., Maloney, M. K., Schmehl, M., Harris, M., Mendez, R., Mendez, R. and Sandford, P. A. Insulin independence in a type I diabetic patient after encapsulated islet transplantation, Lancet 343: 950-951 (1994); Weber, C. J. et al. Xenografts of microencapsulated rat, canine, porcine and human islets, In: Pancreatic Islet Cell Transplantation, Ricordi C, ed., R. G. Landes, Austin, pp. 177-190 1992]. Methods such as interfacial polymerization or chemical crosslinking of polymers use organic solvents or toxic chemicals (which are also toxic to the encapsulated living cells). The technique developed by Lim and Sun, employing an alginate/polylysine membrane system, has been implemented with some success [Lim, F. and Sun, A. M. Microencapsulated islets as bioartificial endocrine pancreas, Science 210: 908-910 (1989)]. However, transplantation with this type of capsule has been limited by fibrosis of the capsules in large animals (resulting in anoxia and death of islets), and a limited life span of the capsules. Clearly, further progress in the design of capsules for immunoisolation of islets and in the facilitation of their survival is needed.
Adverse effects of anoxia can be partially alleviated by the ability of islets to upregulate synthesis of angiogenic factors under hypoxia. It has been shown that the expression of VEGF in isolated islets is upregulated following a period of hypoxia and hypoglycemia in vitro [Gorden, D. L., Mandriota, S. J., Montesano, R., Orci, L., Pepper, M. S. Vascular endothelial growth factor is increased in devascularized rat islets of Langerhans in vitro, Transplantation 63: 436-443 (1997)] and may partially relieve a lack of oxygen in the initial phases of transplant fibrosis. However it cannot achieve a complete abrogation. In addition, this capability may be impaired due to islet damage during the enzymatic digestion process that isolates the islets from the pancreas.
Recent advances in the understanding the molecular mechanisms of wound healing process and of normal and pathological angiogenesis provide solid basis for the clinical application of vascularizing growth factors. Further clinical development is only possible through designing regimens that can practically address the problems of bioavailability.
The prior art is deficient in the lack of polymer encapsulation systems which promote vascularization of tissues surrounding transplanted capsules and which prevent fibrosis from isolating the capsule contents. The present invention fulfills this longstanding need and desire in the art.
The present invention is based on a unique formulation method using multicomponent water-soluble polymers formed into polymeric sheets. This preparation permits modification to a desirable size, provides adequate mechanical strength and exhibits exceptional permeability and surface characteristics.
One embodiment of present invention is a polyanionic/polycationic polymeric composition further comprising a protein which may be an angiogenic stimulating factor, a growth factor, or an extracellular matrix protein. Possible polyanion core components are kappa carrageenan, low-esterified pectin (polygalacturonic acid), polyglutamic acid; CMC, carboxymethylcellulose; ChS-6, chondroitin sulfate-6; ChS-4, chondroitin sulfate-4; F-68, Pluronic, CMC, ChS-6, ChS-4, and collagen. Representative cationic shell components include polyvinylamine, spermine hydrochloride, protamine sulfate, polyethyleneimine, polyethyleneimine-ethoxylated, polyethyleneimine, epichlorhydrin modified, quartenized polyamide and polydiallyldimethyl ammonium chloride-co-acrylamide, and low molecular-weight chitosan. In a preferred embodiment, the polymeric film is synthesized from the polyanions high viscosity sodium alginate and cellulose sulfate and the calcium chloride and poly(methylene-co-guanidine) hydrochloride (PMCG) (polycation). Possible angiogenic stimulating factors include vascular endothelial growth factor (VEGF), angiopoietin (APO), transforming growth factor (TGF) TGFxcex2, acidic fibroblast growth factor (FGF) aFGF, basic fibroblast growth factor bFGF, and the combination of aFGF and bFGF collectively referred to as FGF. Platelet derived growth factor (PDGF) is a possible growth factor, while possible extracellular matrix components include heparin, heparan sulfate, hyaluronic acid, fibronectin, laminin, and perlecan.
It is contemplated that a pharmaceutical composition may be prepared using a drug encapsulated in the said vehicle of the present invention. In such a case, the pharmaceutical composition may comprise a drug (vascularization agent) and a biologically acceptable matrix. A person having ordinary skill in this art readily would be able to determine, without undue experimentation, the appropriate concentrations of said biotechnology products, matrix composition and routes of administration of the vehicle of the present invention.
Another embodiment of the present invention is the encapsulation of animal cells in a polymer containing an angiogenic stimulating factor. The vascularization factor promotes vascularization around the microencapsulated cells to prevent hypoxia of the cells within the microcapsules. In addition, it prevents the formation of fibrous tissue around the microcapsules, resulting in the isolation of the microcapsules. The polymer may also include growth factors in addition to the angiogenic stimulating factor.
In yet another embodiment of the present invention, the area in which the cell are to be implanted may be prevascularized by placing a polymer mesh or capsule containing an angiogenic stimulating factor in the individual for at least 2 weeks prior to implanting encapsulated cells.
Preferably, the animal cells are pancreatic islet cells. The pancreatic islet cells may be implanted as microencapsulated cells. Growth factor may also be incorporated in the polymer encapsulating the cells or may be present in polymeric films, microcapsules, or nanoparticles encapsulated along with the islet cells. For example, microencapsulated pancreatic islet cells and nanoparticulate FGF may be implanted together in a capsule in the peritoneum of the kidney. To slow the release of the growth factor, polydextran aldehyde may be used to crosslink the growth factor to the polymer.
In another embodiment of the present invention, a method is provided for accelerating in vivo wound healing by placing a polymeric composition containing growth factor in proximity to a wound. Platelet derived growth factor would be especially efficacious in promoting wound healing. Alternatively, the form of the polymer may be a polymeric film containing FGF, nanoparticle-FGF, or FGF-hydrogel-coated (and possibly crosslinked with polydextran aldehyde) bioresorbable film for wound healing. In additional, The FGF can be crosslinked to the polymer via via Schiff-base polydextran aldehyde complex to slow release of the growth factor.
In another embodiment of the current invention, a dialysis based method of making polymeric sheets for drug and cell encapsulation is described, including the following steps: (1) introduction of a solution of polyanionic monomers into a sterile dialysis cassette; (2) immersion of said cassette in a stirred reactor containing a polycationic shell solution, (3) diffusion of the polycationic solution inside the cassette to form a polymeric film. The reaction may proceed for few minutes to few hours. For many combinations of polymers, the initial formation of film is observed within few minutes. However considerable time is required to mature into mechanically self-sustainable films. The resulting polymeric film has an excess of positive charge on the outside of the film which may be neutralized by incubation with a dilute polyanionic solution. Proteins may be incorporated in the resulting polymer by adding the protein to the polyanion solution during dialysis. The said composition can be used for production of polymeric films, microcapsules and nanoparticles.
Yet another embodiment of the present invention is the coating of prefabricated structures with a polymer containing a desired protein in a coating process involving sequential application of polyanion and polycation coats. The protein in question is included in the polyanion coat.
A further embodiment is a three-dimensional matrix which provides a resident structure for microencapsulated pancreatic islet cells. Careful selection of the polymers forming the three-dimensional structure permits the slow release of an embedded vascularization agent. In this manner, ingrowth of blood capillaries towards the encapsulated pancreatic islets is encouraged. Both polymers and agent encourage capillary network development and inhibit or eliminate growth of the dense and impermeable fibrotic cellular structures that are often associated with implant failure.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.
As appearing herein, the following terms shall have the definitions set out below.
As used herein, the term xe2x80x9cdrugxe2x80x9d shall refer to a chemical entity of varying molecular size (both small and large) exhibiting a therapeutic effect in animals and humans.
As used herein, the term xe2x80x9creactorxe2x80x9d refers to an enclosed vessel provided with or without stirrer, allowing a reaction to proceed in liquid or gas phases.
As used herein, the term xe2x80x9cfilm vehiclexe2x80x9d shall refer to a microscopic gelled solid object of slab geometry.
As used herein, the term xe2x80x9cmicrocapsulexe2x80x9d shall refer to microscopic (few micrometers in size to few millimeters) solid object, essentially of regular spherical shape, exhibiting a liquid core and a semipermeable shell.
As used herein, the term xe2x80x9cnanoparticlexe2x80x9d shall refer to submicroscopic (less than 1 micrometer in size) solid object, essentially of regular or semi-regular shape.
xe2x80x9cAs used herein, the term xe2x80x9cshellxe2x80x9d refers to an insoluble polymeric electrostatic complex composed of internal core polymer(s) and external bath polymer(s) molecularly bonded (gelled) in close proximity.
As used herein, the term xe2x80x9cstructural (gelling) polymerxe2x80x9d shall refer to polymers, which can form semi-solid gelled structures by means of small ion complexing.
As used herein, the term xe2x80x9ccore polymerxe2x80x9d shall refer to an internal part of the microcapsule, nanoparticle or polymeric film.
In the description of the present invention, the following abbreviations may be used: SA-HV, high viscosity sodium alginate; CS, cellulose sulfate; k-carr, kappa carrageenan; LE-PE, low-esterified pectin (polygalacturonic acid); PGA, polyglutamic acid; CMC, carboxymethylcellulose; ChS-6, chondroitin sulfate-6; ChS-4, chondroitin sulfate-4; F-68, Pluronic copolymer; PVA, polyvinylamine; 3PP, pantasodium tripolyphosphate; PMCG, poly(methylene-co-guanidine) hydrochloride; SH, spermine hydrochloride; PS, protamine sulfate; PEI, polyethyleneimine; PEI-eth, polyethyleneimine-ethoxylated; PEI-EM, polyethyleneimine, epichlorhydrin modified; Q-PA, quartenized polyamide; pDADMAC-co-acrylamide, polydiallyldimethyl ammonium chloride-co-acrylamide; PBS, phosphate-buffered saline; ECM, extracellular matrix molecule; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; TGFxcex2, transforming growth factor xcex2; APO, angiopoietin.
The present invention is directed to a composition of matter comprising various polyanionic/polycationic polymer compositions incorporating angiogenic stimulating factors, growth factors, or extracellular matrix proteins. Among useful polyanions for making polymeric films, capsules and nanoparticles are k-carr, LE-PE, PGA, CMC, ChS-6, ChS-4, and collagen. Possible polycations include PVA, SH, PS, PEI, PEI-eth, PEI-EM, Q-PA and pDADMAC-co-acrylamide, and low molecular-weight chitosan, among others.
The present invention can be used to stimulate vascularization around a polymeric capsule which may include a drug or microencapsulated cells. Among useful angiogenic stimulating growth factors for this purpose are VEGF, aFGF and bFGF collectively referred to as FGF, APO, and TGFxcex2.
An area may be prevascularized by placing a retrievable polymeric mesh or capsule incorporating an angiogenic stimulating factor in an individual for at least 2 weeks.
The instant invention is especially useful for the implantation of microencapsulated pancreatic islet cells. A growth factor may also be included in either the polymer encapsulating the cells or in a separate polymeric composition to stimulate the growth of the pancreatic islet cells. Release of the growth factor may be slowed by crosslinking the growth factor to the polymer with polydextran aldehyde. The encapsulated pancreatic islet cells and growth factor may be implanted in a capsule in the peritoneum of the kidney.
In addition, the present invention describes a method of accelerating in vivo wound healing by placing the polymeric vehicles containing an appropriate angiogenic growth factor in proximity to a wound. Representative growth factors include platelet derived growth factor and fibroblast growth factors. Release of the growth factors may be slowed by crosslinking the factors to the polymer with polydextran aldehyde. The present invention provides a dialysis process for making sterile polyanionic/polycationic polymeric compositions. A particularly usable combination is anionically charged alginate/CS, cationically charged PMCG/calcium chloride. Proteins such as angiogenic growth factors may be incorporated into these compositions by including them in the polyanion mixture during dialysis. Even if a protein is cationically charged (as is the case of bFGF), it is still incorporated into the multicomponent polyanionic mixture, as the formed electrostatic complex between the anions and such growth factor is water soluble at the concentrations of angiogenic growth factor used.
Additionally, the method of coating of prefabricated polymeric films and other three-dimensional structures with the polyanion/polycation polymeric composition is described. This is done by a sequential coating process where polyanion and polycation coats are alternatively applied. The material can be coated with protein factors by including these factors in the polyanion coat.
Effective treatment of medical conditions in the human body often requires the creation or restoration of blood vessels. This vascularization, process can be the crucial step in therapies for highly dissimilar ailments, and its failure can mean the difference between long-term success and a brief improvement followed by decline or even death. Furthermore, the value of a newly-formed capillary subsystem can be undermined if a fibrous tissue formation occurs. These issues are addressed herein for the case of diabetes.
A three-dimensional matrix is suggested in this invention which provides a resident structure for microencapsulated pancreatic islet cells. Careful selection of the polymers forming the three-dimensional structure permits the slow release of an embedded vascularization agent. In this manner, ingrowth of blood capillaries towards the encapsulated pancreatic islets is encouraged. Both polymers and agent encourage capillary network development and inhibit or eliminate growth of the thick fibrotic cellular structures that are often associated with implant failure.
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.